Enzymatic-electrochemical one-shot affinity sensor for the quantitative determination of analytes for aqueous media and affinity assay

ABSTRACT

The invention relates to an enzymatic-electrochemical affinity sensor and a one-step affinity assay for the quantitative determination of analytes in aqueous media. More specifically, the invention relates to an enzymatic-electrochemical signal amplification system for a highly sensitive indication of affinity reactions and is particularly suitable in the form of a one-step affinity sensor for in situ analytics. The invention is also directed to the use of phenol oxidase as a marker enzyme for the affine binding partners in an electrochemical affinity sensor or assay, and to the use of an enzyme hydrolyzing phenolic compounds as marker enzyme for the affine binding partners, in combination with a phenol oxidase as catalyst for the amplifying reaction in an electrochemical affinity assay.

The invention relates to an enzymatic-electrochemical affinity sensorand a one-step affinity assay for the quantitative determination ofanalytes in aqueous media. More specifically, the invention relates toan enzymatic-electrochemical signal amplification system for a highlysensitive indication of affinity reactions and is particularly suitablein the form of a one-step affinity sensor for in situ analytics. Theinvention is also directed to the use of phenol oxidase as a markerenzyme for the affine binding partners in an electrochemical affinitysensor or assay, and to the use of an enzyme hydrolyzing phenoliccompounds as marker enzyme for the affine binding partners, incombination with a phenol oxidase as catalyst for the amplifyingreaction in an electrochemical affinity assay.

In order to detect immunochemical reactions or affinity reactions ingeneral, a number of electrochemical or enzymatic-electrochemicalindication systems are known which are based on the indication of anelectrochemically active marker (Heinemann and Halsall, Anal. Chem. 57(1985), pp. 1321A-1331A; Patent Specification DE 42 16 696; Le Gal LaSalle, J. Electroanal. Chem. 350 (1993), 329-335) or anelectrochemically active product of the marker enzyme. Above all,alkaline phosphatase (Doyle et al., Anal. Chem. 56 (1984), pp.2355-2360; McNeil et al., Biosensors 3 (1987), pp. 199-209; Duan et al.,Anal. Chem. 66 (1995), pp. 1369-1377; Meusel et al. Biosens. &Bioelectron. 10 (1995), pp. 577-586) or galactosidase (Masson et al.,Anal. Chim. Acta 304 (1995), pp. 353-359) is used as marker enzyme. Avariety of other enzymatic-electrochemical indication systems are basedon the cyclic regeneration of redox-active reactants.

The most frequently described assays are those where either a redoxenzyme or a redox mediator is used as a marker for an antigen or anantibody. Following the corresponding immunochemical reaction, anenzymatic redox reaction sequence is completed by the marker in thepresence of the enzyme substrate, wherein a redox mediator, aredox-active prosthetic group of the enzyme, or a redox-active enzymaticco-factor, or a redox-active co-substrate is reduced or oxidized. Usingan amperometric redox electrode, the redox system is regenerateddirectly or via a mediator. The current resulting therefrom depends onthe analyte concentration, i.e., the redox-active conjugates which,depending on the analyte, is provided by the immune reaction at just alow concentration, causes a cyclic enzymatic-electrochemicalregeneration reaction resulting in an amplified signal generation, witha correspondingly high measurement current. The redox marker may be acomponent of a homogeneous or a heterogeneous immunoassay or animmunoassay according to the competition, titration or displacementprinciple.

Weber and Purdy (Anal. Letters 12 (1979), pp. 1-9) have been the firstto accomplish a homogeneous immunoassay using ferrocene as redox-activelabel of an antigen and detect the direct oxidation of the ferroceneconjugate at −500 mV (vs. SCE).

One enzymatic-electrochemical immunoassay (diGleria et al., Anal. Chem.58 (1986), pp. 1203-1206) also uses ferrocene as antigen label, whereinglucose oxidase is used in the mediated indicator reaction of theantigen-ferrocene conjugate which is displaced from the binding site ofthe antibody in the presence of analyte and thus, may assume theelectron transfer between said oxidase and the electrode.

The perfection of the above enzymatic-electrochemical immunoassays hasbeen described in the Patent Specification EP 125,139. Again, a redoxmediator is used as label for the antigen or the antibody. In thepublication by Suzawa et al. (Anal. Chem. 66 (1994), pp. 3889-3894),ferrocene is used as multi-label in combination with glucose oxidase.Another well-known immunoassay (Gyss and Bourdillon, Anal. Chem. 59(1987), pp. 2350-2355) uses glucose oxidase as marker, whereinbenzoquinone serves as mediator.

Increasing the sensitivity of amperometric indication systems on thebasis of redox enzyme/mediator sequences in immunoassays is the aim ofPatent Specification EP 241,309. Therein, a second electron acceptor(ferricyanide, polyvinylferrocene or Berlin blue) is introduced into themeasuring solution or used to modify the electrode surface andaccumulate reduction equivalents from the enzymatic glucose oxidationthe via hapten-ferrocene conjugate. Following an accumulation period,the amperometric measurement of the electron acceptor is effected, whichhas been reduced during this accumulation phase.

In the Patent Specification EP 223,541, use is made of redox mediatorsshifted in negative direction in their formal potential by coupling aphosphate group or a phenol derivative so that, in contrast to theirnon-derivatized form, no electron transfer from glucose oxidase to theelectrode surface via mediator can occur. Ferrocene ordichlorophenol-indophenol are used as mediators. In the presence of ahapten conjugate which has alkaline phosphatase as marker enzyme andresults from a competitive reaction with the analyte, cleavage of thederivatizing group occurs so that the mediator may assume the electrontransfer between glucose oxidase and the redox electrode. Depending onthe concentration of the available marker enzyme-antigen/antibodyconjugate, an anodic measurement current will occur.

Furthermore, a measuring system is known (PCT 86/03837) wherein themarker enzyme itself does not catalyze the indicative redox reaction butrather, generates a redox-active “trigger substance” which either may bedetected directly by amperometry or completes anenzymatic-electrochemical amplification sequence as a result of itsreversible redox behavior. Alkaline phosphatase or β-galactosidase isused as marker enzyme. In the event of alkaline phosphatase, NADP⁺ isused as “trigger substrate” which is hydrolyzed to NAD⁺ by eliminationof a phosphate group and, as a co-factor in an ethanol/alcoholdehydrogenase/diaphorase, ethanol/alcohol dehydrogenase/ferricyanide, orethanol/alcohol dehydrogenase/ferrocene/ferricyanide redox electrodesequence, results in the completion of this redox cascade. In analogy,electrochemical-enzymatic assays on the basis of the direct ormediator-coupled indication of the NADH co-factor have been described byEggers et al. (Clin. Chem. 28/9 (1982), pp. 1848-1851) and Cardosi etal. (Electroanalysis 1 (1989), 297-304). Alternatively, the alkalinephosphatase marker enzyme may be used to cleave the phosphate group ofan SH-containing compound which then acts as an electron donor for aglutathione reductase or diaphorase and is regenerated cathodically. Inaddition, this Patent Specification also describes the use ofβ-galactosidase as marker enzyme hydrolyzing thep-hydroxyphenyl-β-galactoside. The p-hydroxyquinone having formed servesas substrate for laccase and again, is regenerated cathodically, so thatthe resulting reduction current is proportional to the concentration ofthe anti-body/enzyme conjugate.

An enzymatic electrochemical indication system for immunochemicalreactions is known from Patent Specification PCT 86/04926, which systemhas a depolymerase/ligand conjugate and a redox sequence consisting ofan oxidoreductase, a mediator and a redox electrode spatially separatedfrom each other by a polymer, or which contains one of theabove-mentioned redox components bound in a polymer or incorporated in apolymer. Preferably, a polysaccharide or a liposome matrix is used aspolymer, amyloglucosidase, α-amylase or phospholipase is used asdepolymerase, ferrocene or a ferrocene derivative is used as mediator,and glucose oxidase or glucose dehydrogenase is used as redox enzyme.

The depolymerase used as marker enzyme causes cleavage of monomers frome polymer, which serve as substrates for an oxidoreductase or becomediffusible in the form of mediator-coupled monomers so that in eithercase, an enzymatic-electrochemical reaction is generated. Other possibledepolymerase reactions result in liberation of the redox enzyme enclosedin liposomes, or the penetration of a polymer membrane previously havingseparated the mediator from the redox enzyme. Again, the amperometriccurrent resulting from this redox sequence is proportional to the markerenzyme-ligand conjugate concentration.

Finally, peroxidase-coupled electrochemical-enzymatic immunoassays havebeen described. A well-known homogeneous electrochemical-enzymaticimmunoassay (J. P. O'Daly et al., Enzyme Microb. Technol. 14 (1992) 4,pp. 299-302) uses a ferrocene derivative as label for a hapten whichinitially is electrochemically inactive as a result of binding of theconjugate to the antibody. In the presence of the analyte, adisplacement reaction occurs, and the mediator used as hapten labelcompletes a peroxidase-catalyzed redox reaction generating a reductioncurrent which is proportional to the analyte concentration. In a reverseprocedure (Pritchard et al., Anal. Chim. Acta 310 (1995) pp. 251-256), aperoxidase has been used to label antibodies, the label being detectedcathodically via ferrocenemonocarboxylic acid according to a multi-stepprocedure. For a disposable immunosensor used to detect a low molecularweight analyte in a competitive multi-step assay (Kàlab and Sklàdar,Anal. Chim. Acta 304 (1995), pp. 361-368), a peroxidase has been used asenzyme label of the antibody conjugate which, following a competitivereaction within a defined incubation period between the analyte and thehapten immobilized on the electrode surface, and a washing step, isdetected at the electrode using hydroguinone. In another well-knownpublication (Wright et al., Biosens. & Bioelectron. 10 (1995), pp.495-500), glucose oxidase is used as enzyme label, the reaction productof which (hydrogen peroxide) being generated in the actual indicationreaction via a mediator-free peroxidase-carbon electrode produced usingscreen printing. However, these well-known electrochemical-enzymaticindication systems for immunochemical reactions or affinity reactionssuffer from the general drawback of being either excessively expensiveor insufficiently sensitive for practical applications.

The invention was therefore based on the object of providing an affinityassay featuring high sensitivity, easy handling, and an electricalquantification of the measured signal. More specifically, it should besuitable in the form of a one-step affinity sensor in the in situanalytics in aqueous media.

According to the invention, said object is accomplished by means of anenzymatic-electrochemical one-step affinity sensor in accordance withclaim 1, the preferred embodiments of subclaims 2 through 18, and bymeans of an affinity assay according to claim 19, and the associatedembodiments of subclaims 20 through 25.

In particular, the sensor or assay according to the invention issuitable to detect low molecular weight substances such as pesticides,PCAHs, PCBs, chlorophenols, heavy metal ions, pharmaceuticals, and highmolecular weight substances such as peptides, hormones, proteins,nucleic acids, glycoproteins, cell messengers, as well as microorganismsand viruses.

Hence, the invention is particularly useful in the fields ofenvironmental analytics, medical diagnostics, food analytics, and thecontrol of biotechnological processes.

In the meaning of the invention, ligand or receptor mean the affinebinding partners, e.g., antigen and antibody in the event of animmunoassay. The enzyme-labelled binding partners are denoted asconjugates, and the enzymes as marker enzymes. The complexes betweenligands and receptors will be referred to as affinity complexes, or ascomplexed conjugates if one of the affine binding partners of thecomplex is enzyme-labelled.

The signal-amplifying enzymatic-electrochemical system for detectingaffinity reactions, which is the essence of the invention, is based onthe use of either a ligand labelled with phenol oxidase, which ligandwill undergo an affine reaction with receptors, or a receptor labelledwith phenol oxidase, which receptor will undergo an affine reaction withmodified ligands or the analyte. The phenol oxidase-receptor conjugateor phenol oxidase-ligand conjugate not bound to an immobilized receptoror ligand during the competitive reaction or displaced during thecompetitive reaction, or the complex formed upon a pseudo-homogeneousbinding reaction between the analyte and the phenol oxidase-receptorconjugate will diffuse through solid phases arranged one after another,which consist of diffusible or immobilized phenol oxidase-ligandconjugate or diffusible or immobilized phenol oxidase-receptor conjugateand succeeding diffusion barriers, to a spatially remote redox electrode(3). In the immediate vicinity of the surface, a previously storedenzyme substrate of the phenol oxidase is oxidized by the phenol oxidaseof the conjugate or the complexed conjugate to form an electricallyactive product which is reduced cathodically via the reversibly reducedelectron mediator to form a starting substrate of the phenol oxidase.The resulting cyclic substrate regeneration provides a chemical,analyte-proportional current signal amplification which may bequantified using voltammetric methods.

In the case of a hydrolyzing enzyme used as marker enzyme in ananalogous fashion, preferably a phosphatase or a galactosidase, phenoloxidase is fixed as a layer 21 in the immediate vicinity of theelectrode surface, and an educt resulting from hydrolysis of a hydrolasesubstrate previously stored in layer 20 is oxidized by the phenoloxidase used as catalytic layer to form an electrically active productand, again via reversibly reduced electron mediator, reducedcathodically to form a starting substrate of the phenol oxidase. Theresulting cyclic substrate regeneration provides a chemical,analyte-proportional current signal amplification which may bequantified using voltammetric methods.

In the meaning of the invention, monoclonal or polyclonal antibodies,antibody fragments, lectins, protein A and G, nucleic acids, biologicalreceptors or mixtures thereof are used as receptors. Affine haptens,peptides, heavy metal ion complexes, nucleic acids, carbohydrates,proteins, microorganisms, viruses, or fragments of microorganisms orviruses are used as ligands versus the receptor.

Tyrosinase is preferably used as phenol oxidase. Alkaline phosphatase isparticularly preferred as hydrolyzing enzyme. Preferably, phenol,m-cresol, p-cresol, 2,4-xylenol, p-chlorophenol, or catechol aresuitable enzyme substrates of the phenol oxidase, which result ingeneration of a strong signal.

Quinoid redox dyes, quinones, redox-active complex compounds of iron,ruthenium, osmium, cobalt or tungsten, metallocenes, phthalo(yanines, orelectrically conductive redox polymers such as polyaniline, polypyrrole,poly-o-phenylenediamine, or polyacetylene are used as electron mediatorsin the enzymatic electrochemical indication reaction.

The displacement principle is utilized in a first embodiment accordingto the invention. It is based on the use of either a phenoloxidase-ligand conjugate or a phenol oxidase-receptor conjugate whichserves to saturate the binding sites of a corresponding immobilizedreceptor or immobilized ligand wherein the phenol oxidase is conjugatedwith a ligand which, compared to the actual analyte, has a substantiallylower affinity versus the receptor, or a ligand is used forimmobilization to which the phenol oxidase-labelled receptor has asubstantially lower affinity compared to the actual analyte. A moleculehaving a structural relationship to the analyte is used as ligand. Inthe presence of analyte, the phenol oxidase-ligand conjugate isdisplaced by same from the binding sites of the immobilized receptor, orthe immobilized ligand is displaced by the analyte from the bindingsites of the receptor conjugated with phenol oxidase. The displacedphenol oxidase-ligand conjugate or the phenol oxidase-receptor conjugatecomplexed with analyte will diffuse to the spatially remote electrodesurface and undergo a signal-amplifying enzymatic-electrochemical redoxreaction in the presence of a phenol oxidase substrate and a redoxmediator, which reaction generates an analyte-proportional, voltammetrictest signal.

A second embodiment according to the invention is based on a(quasi)homogeneous affinity reaction. In local separation from animmobilized modified ligand, an affine reaction between the analyte andthe phenol oxidase-conjugated receptor present in excess will initiallyoccur. When diffusing to the spatially remote redox electrode, theanalyte molecules completed with the phenol oxidase-conjugated receptor,and the excess phenol oxidase-conjugated receptor will pass a sectionhaving immobilized ligand to which the non-complexed fraction is bound,so that only the analyte-complexed phenol oxidase-conjugated receptorfraction will undergo a signal-amplifying enzymatic-electrochemicalredox reaction at the electrode surface in the presence of a phenoloxidase substrate and a redox mediator.

A third embodiment according to the invention implies an initial affinecompetitive reaction between the analyte and the phenol oxidase-ligandconjugate for the binding sites of an immobilized receptor. The fractionof phenol oxidase-ligand conjugate which has not been bound as a resultof analyte complexing with the receptor will diffuse to the spatiallyremote redox electrode and undergo a signal-amplifyingenzymatic-electrochemical redox reaction in the presence of a phenoloxidase substrate and a redox mediator.

Instead of the phenol oxidase in the above-described embodimentsaccording to the invention, a fourth, particularly preferred embodimentof the invention provides a hydrolyzing enzyme as marker enzyme,preferably a phosphatase or a galactosidase. The phenol oxidase,however, is immobilized directly on the redox electrode surface in theform of a catalytically active layer which then, in the presence of aredox mediator, is capable of introducing the hydrolase used as markerenzyme into the enzymatic-electrochemical detection in asignal-amplifying fashion via one of its products of hydrolysis whichalso is an efficient enzyme substrate of the phenol oxidase.

Ultimately, the phenol oxidase used as marker enzyme is utilized eitherdirectly or as a mediating catalytic layer in the presence of a suitableenzyme substrate and a redox mediator in the actual indication reactionat a cathodically polarized redox electrode wherein the phenol oxidasereacts specific enzyme substrates, preferably phenol, m-cresol,p-cresol, 2,4-xylenol, p-chlorophenol, or catechol to form anelectrochemically active product which in turn is reducedvoltammetrically to a starting substrate for the phenol oxidase by meansof a reversible reduced quinoid redox dye, a quinone, a redox-activemetal complex or an electrically conductive polymer. That is to say, theregenerated starting substrate is also available for the enzymaticoxidation reaction. Surprisingly, such a cyclic substrate regenerationor chemical signal amplification results in a detection limit which,compared to well-known amperometric detection systems, is decreased byabout three orders of magnitude.

The enzymatic-electrochemical one-step affinity sensor according to theinvention consists either of a voltammetric measuring chain 22 printedon a planar support strip 1, or of two identical voltammetric measuringchains 22, 23 printed in close proximity on a common support strip,which chains in either design are covered with a sequence of layers eachimpregnated with various reagents required for the affinity assayaccording to the invention. In the twin embodiment, one of the twopartial sensors on the common support substrate serves as the actualindication system, while the other partial sensor serves as a functionaltest.

Each of the voltammetric measuring chains of the sensors consists of aredox electrode 3 and an Ag/AgCl pseudo-reference electrode 4surrounding same, the surface of said redox electrode being modified bya quinoid redox dye, a quinone, a redox-active complex compound of iron,ruthenium, cobalt, osmium, manganese, or tungsten, a metallocene, aphthalocyanine, or an electrically conductive redox polymer such aspolyaniline, polypyrrole, poly-o-phenylenediamine, or polyacetylene. Theelectrodes, including their contact paths 2 for connection to apotentiostate or a manual measuring instrument, and the necessaryisolation layer 5 between the contact paths are applied to the planarsupport strip using screen printing. The modified redox electrode hascathode polarization versus the pseudo-reference electrode.

Directly above the electrodes, multiple consecutive layers are pressedin close contact onto the electrodes by means of a flexible fixing frame6, 7 made of plastic, which electrodes, in a well-aimed fashion, promotediffusion or capillary forces or inhibit same for a limited period oftime by using appropriate porous materials or modifying same.Preferably, cellulose, polysilicates, linear crosslinked hydrogels or amixture of said materials, optionally provided with additionalhydrophilic components, preferably with a polysaccharide, polyalcohol,poly(ether alcohol), or an inorganic salt, are used as materialspromoting diffusion or capillary forces, which materials are alsoprovided with appropriate buffer substances. Hydrophobized paper ispreferably used as diffusion barrier layer which is to act as such for alimited period of time. In a preferred embodiment, the areas of thelayers towards the electrode surface are of a design so as to decreasein order to achieve an efficient accumulation effect.

Towards the measuring medium, the layers are partially covered by awater-impermeable film or membrane 9, preferably made of PTFE,polycarbonate or a rubber compound, which is spaced apart from the cover8 of a fixing frame by means of an annular porous spacer 10. The fixingframe 6, 7 which includes a cylindrical cavity to accommodate the layersand protects them against lateral penetration of measuring medium has acircular area in its cover 8. Said area preferably has regularlyarranged openings to allow entry of the measuring medium, and is smallerin diameter than the film. Immediately following the film which in turnhas a smaller diameter than the cylindrical cavity of the fixing frame,a sample-receiving and reservoir layer 11 is arranged, consisting of amaterial which rapidly absorbs water and has good swellability,preferably a cellulose layer impregnated with a natural or synthetichydrogel such as agar, gelatin, pectin, dextrin, polyacrylamide, orpolyurethane. When applying an aqueous sample, the sample will diffusethrough the openings of the fixing frame cover 8 and the annular spacer10 into the sample-receiving and reservoir layer 11 until the latter, asa result of its swelling and the volume increase associated therewith,will press the film 9 against the inlet openings of the fixing framecover 8, so that further supply of sample is prevented. In this fashion,a well-defined sample volume is provided.

The sample-receiving and reservoir layer 11 is followed by a layer 12which either is provided with freely diffusible phenol oxidase-receptorconjugate or phenol oxidase-conjugated modified ligand and is followedby a diffusion-inhibiting layer 13.

In the event cf the phenol oxidase-receptor conjugate present indiffusible form, an affine reaction with the analyte occurs, while inthe event of the phenol oxidase-ligand conjugate present in diffusibleform, the aim merely is homogeneous distribution of the analyte withinsaid layer. In either event, the incubation period will be determined bythe period of time required to break through the succeeding diffusionbarrier 13.

According to either of these different cases, the next layer 14 containseither a ligand or a receptor immobilized on the solid phase, whichlayer in turn is followed by a diffusion barrier layer 15. In the firstcase, the fraction of phenol oxidase-receptor conjugate which failed tobind analyte in the preceding affine reaction will be bound to theimmobilized ligand, while the phenol oxidase-receptor conjugatecomplexed with analyte will diffuse through said layer as soon as thediffusion barrier is penetrated. In the second case, the analyte and thephenol oxidase-ligand conjugate compete for the binding sites of theimmobilized receptor. As a result of the higher affinity of the analyteand the limited amount of receptor, the excess fraction of phenoloxidase-ligand conjugate will diffuse further in an analyte-proportionalfashion after breaking through the diffusion barrier 15.

In the following last layer 16, either a receptor for the phenoloxidase-receptor conjugate complexed with analyte or a receptor for thephenol oxidase-ligand conjugate is immobilized on the solid phase foraccumulation. Said layer 16 is located immediately in front of theelectrode surface and is surrounded by a circular layer 18 which isseparated by a diffusion barrier 17 and contains the phenol oxidasesubstrate. Following an incubation period required for accumulatingeither the phenol oxidase-receptor conjugate complexed with analyte orthe phenol oxidase-ligand conjugate complexed with analyte thesubstrate, after breaking through the diffusion barrier 17, willpenetrate into layer 16 in the vicinity of the electrode, and the actualenzymatic-electrochemical indication reaction will take place at thecathodically polarized redox electrode 3 via the redox mediator bound byadsorption, physical occlusion or covalently to the electrode surface 3.

A second embodiment of the enzymatic-electrochemical one-step affinitysensor according to the invention has a layer 19 immediately followingthe sample-receiving and reservoir layer 11, which includes a receptoror modified ligand immobilized on the solid phase and complexed eitherwith phenol oxidase-ligand conjugate or phenol oxidase-receptorconjugate. Following diffusion of the sample fluid out of thesample-receiving and sample reservoir layer 11, a displacement reactioncaused by the analyte takes place, so that either the analyte will bindto the immobilized receptor, displacing the phenol oxidase-ligandconjugate, or the analyte will complex with the phenol oxidase-receptorconjugate previously bound to the immobilized ligand. Again, afterbreaking through the diffusion barrier 13, the diffusible fraction ofeither the phenol oxidase-ligand conjugate or the analyte-complexedphenol oxidase-receptor conjugate resulting from the above will diffuseinto the layer 16 in the vicinity of the electrode, which layer isdesigned as already described above.

A third embodiment of the enzymatic electrochemical one-step affinitysensor according to the invention implies the use of a hydrolyzingenzyme as marker enzyme instead of phenol oxidase and, in contrast tothe above-described embodiments, has an additional phenol oxidase layer21 as layer in the vicinity of the electrode, which is directlyimmobilized on the electrode surface.

The advantages achieved using the enzymatic-electrochemical affinityassay and the associated enzymatic-electrochemical one-step affinitysensor are particularly to be seen in the fact that an extremelysensitive, electrically quantifiable detection can be effected,particularly of small analyte molecules as well, the one-step measuringsystem free of reagents and separation enabling easy handling.

Another advantage is provided by using two identical and closelyadjacent partial sensor arrays on the same support substrate. As aresult of the well-defined diffusible analyte concentration which, incontrast to the actual indication array, is contained in thesample-receiving and reservoir layer 11 of the functional test array,the cathodic measured current of this partial sensor will be higher by adefined difference compared to the indication partial sensor. Thus, inaddition to the actual analyte determination, it is possible at the sametime to effect a functional control during the measuring procedure,augmenting the reliability of measurement.

In the following Examples 1-3 concerning the determination of the2,4-dinitrophenol (DNP) model analyte, and in Example 4 concerning thedetermination of IgA, and with reference to FIGS. 1 through 5b, theinvention will be illustrated in more detail without limiting it to theabove.

FIG. 1 shows an embodiment of the enzymatic-electrochemical one-stepaffinity sensor according to the invention in perspective view.

FIG. 2 shows a sectional view along the line A-A′ in FIG. 1 of a variantof the enzymatic-electrochemical one-step affinity sensor of theinvention according to claim 7.

FIG. 3 shows a perspective view of an embodiment of theenzymatic-electrochemical one-step affinity sensor of the inventionhaving a second identical sensor channel on the support to controlcalibration and function.

FIG. 4 shows a sectional view along the line A-A′ in FIG. 1 of a variantof the enzymatic-electrochemical one-step affinity sensor of theinvention according to claim 8.

FIG. 5a shows a sectional view along the line A-A′ in FIG. 1 of avariant of the enzymatic-electrochemical one-step affinity sensor of theinvention according to claim 9.

FIG. 5b shows a sectional view along the line A-A′ in FIG. 1 of avariant of the enzymatic-electrochemical one-step affinity sensor of theinvention according to claim 10.

EXAMPLE 1

(FIGS. 1, 2)

Carbon contact paths 2, an N-methylphenazinium Reineckate-modifiedcarbon working electrode 3, a silver/silver chloride (Ag/AgCl) referenceelectrode 4 surrounding the working electrode 3 in the form of a squareribbon, and an isolating layer 5 which, in addition to the surfaces ofthe working and reference electrodes and plug contact surfaces, coversthe support, are printed consecutively on a glass fiber-epoxide resinsupport 1 using polymer thick-layer pastes and cured at 90° C. Directlyon the measuring window surface where the working electrode 3 and thereference electrode 4 are arranged, a fixing frame 6, 7 made of plasticis clamped by appropriate shapings onto the support 1. The fixing frameincludes a cylindric cavity and has regularly arranged openings 0.1 mmin diameter in its top cover 8 over a limited circular area 1.5 mm indiameter. The fixing frame is tightly packed with a sequence ofdifferent layers: Directly below the perforated circular area of thefixing frame cover 8, there is a Silopren film 9 (ø 3 mm) spaced apartfrom the cover by an annular spacer 10 (ø 3 mm, ø_(i) 2 mm) made offilter paper. The Silopren film has been produced by polycondensationthrough addition of an appropriate catalyst (crosslirker KA-1, Bayer AG)to the Silopren K1000 (Fluka) on a PTFE backing to form a film, andpunched out in the form of small plates. The Silopren film is followedby a swellable layer 11 having a diameter of 5 mm, which consists of afilter paper coated with SANWET® IM 3900 G (Hoechst AG) and serves assample-receiving and reservoir layer.

This layer, as well as the next filter paper layers were prepared usingBlauband round filter paper (Schleicher & Schull 589/3) previouslyimpregnated with an aqueous solution of 2 vol.-% dextran, m.w. 70,000(Sigma), 10 vol.-% glycerol, and 0.1 M phosphate buffer, pH 6.8.

The round filter paper had a diameter of 55 mm and was punched outmatching to the respective diameter of the cylindrical cavity of thefixing frame (5 mm-1 mm), according to the respective modificationprocedures.

The sample-receiving and reservoir layer is followed by a filter paperlayer 12 (ø 3 mm) containing freely diffusible DNP-L-lysine-tyrosinaseconjugate. The ε-2,4-DNP-L-lysine (Sigma) was coupled to tyrosinase,2000 U/mg (Sigma) in analogy to the mixed anhydride method according toJung et al., J. Agric. Food Chem. 37 (1989), 1183. Directly below, aslightly hydrophobized paper layer 13 (ø 3 mm) is arranged as diffusionbarrier, followed by a filter paper layer 14 (ø 1 mm) having scavengerantibodies immobilized in a directed fashion. To prepare thelast-mentioned layer, the filter paper was initially subjected to ahydrolysis procedure. The resulting OH groups were activated usingcarbonyldiimidazole and covalently bound to the carbohydrates of the Fcportion of the antibodies (2 ml) previously oxidized with periodate,using succinic acid ddhydrazide. For preparation, the well-knownprocedures were used, such as described in M. B. Wilson and P. K.Nakane: “Immunofluorescence and Related Staining Techniques”, Elsevier,North Holland Biomedical Press, pp. 215-224; and in G. T. Eermanson, A.K. Mallia and P. K. Smith: Immobilized Affinity Ligand Techniques,Academic Press, San Diego, Calif., 1992. Commercially availablepolyclonal anti-DNP antibodies from rabbits (Sigma) were used asantibodies. In order to minimize non-specific binding, theimmobilization matrix was blocked with a 1% casein-phosphate buffer/KClsolution.

Again, this is followed by a hydrophobized paper layer 15 (ø 1 mm) andonce more by the same filter paper layer 16 (ø 1 mm) having immobilizedantibodies, which comes to lie directly on the surface of the workingelectrode. Said filter paper layer 16 simultaneously forms the space inclose vicinity to the electrode and is surrounded by a filter paper ring18 (ø 2 mm, ø_(i) 1 mm) impregnated with 0.1 mM catechol, the innerdiameter of which is impregnated with silicone, thereby creating adiffusion barrier 17.

When applying a drop of aqueous sample to the perforated fixing framecover 8, the sample fluid will first diffuse into the sample-receivingand reservoir layer 11 via spacer 9. As a result of swelling of thesample-receiving and reservoir layer 11 caused in this way, the Siloprenfilm 9 is pressed against the Perforated fixing frame cover 8, thusclosing same. The aqueous sample diffuses through the succeeding filterpaper layer 12 containing dinitrophenol-tyrosinase conjugate, mobilizingthe conjugate. After breaking through the hydrophobized paper layer 13serving as diffusion barrier, both the conjugate and the analyte enteranother filter paper layer 14 containing the scavenger antibodyimmobilized therein. During the heterogeneous immune reaction, thehapten conjugated with tyrosinase and the dinitrophenol will compete forthe binding sites of the antibodies immobilized in a directed fashion.Owing to the higher affinity of the analyte molecules versus theconjugate, complexing of the analyte is preferred. The more analyte ispresent in the sample, the less DNP-L-lysine-tyrosinase conjugate willbe bound. A sufficient incubation period for the competitive immunereaction is ensured by the hydrophobic paper layer 15 arranged insuccession. Having overcome this diffusion barrier, the non-boundhapten-enzyme conjugate enters the layer 16 in the vicinity of theelectrode, which in turn is provided with immobilized antibodies againstDNP, and is accumulated directly in front of the electrode surface 3 viabinding to said antibodies. Because the sample is an aqueous one, thecatechol used as enzyme substrate will be dissolved from the filterpaper ring 18 surrounding the layer in the vicinity of the electrode,initiating its enzyme-catalyzed reaction. The catechol is oxidized too-quinone which is reduced back to catechol by the cathodically reducedN-methylphenazinium on the electrode surface 3. The cathodic measuredcurrent resulting from this cyclic enzymatic-electrochemical substrateregeneration, which is detected as peak current using square wavevoltammetry at −160 mV versus internal Ag/AgCl pseudo-referenceelectrode 4, is proportional to the DNP concentration. The measuringrange of this one-step immunosensor is between 0.5 and 20 μg/l for DNP.

EXAMPLE 2

(FIG. 5a)

The second embodiment is based on the same sensor design as described inExample 1, but uses alkaline phosphatase at 150 U/mg (Sigma) to labelthe hapten (DNP-L-lysine). Accordingly, the filter paper ring 20 isimpregnated with 1 mM phenyl phosphate, and an additional layer 21containing tyrosinase, 2000 U/mg (Sigma) immobilized in a PUR hydrogellayer (Kotte et al., Anal. Chem. 67 (1995), 65), is inserted between thelayer 16 in the vicinity of the electrode and the working electrodesurface 3. The immunochemical reaction is analogous to Example 1, theactual indication reaction taking place via the alkalinephosphatase-tyrosinase two-enzyme sequence: The non-boundphosphatase-DNP-L-lysine conjugate which enters the layer 16 in thevicinity of the electrode and is bound by the anti-DNP-antibodiesimmobilized therein, thus being accumulated, will hydrolyze the phenylphosphate diffusing out of the surrounding filter paper ring to formphenol. Eventually, the phenol is oxidized by the following tyrosinaselayer to form o-quinone which is reduced to catechol by the catholicallyreduced N-methylphenazinium on the electrode surface 3. The cathodicmeasured current resulting from this cyclic enzymatic-electrochemicalsubstrate regeneration, which is detected as peak current using squarewave voltammetry at −160 mV versus internal Ag/AgCl pseudo-referenceelectrode 4, is proportional to the DNP concentration. The measuringrange of this one-step immunosensor is between 0.1 and 10 μg/l for DNP.

EXAMPLE 3

(FIG. 3)

This example describes the use of two immunosensor systems 22, 23 whichare arranged in succession on a support and designed as in Example 1.The actual indication system 22 is constituted by one of the “partialsensors” which function independently, while the other one serves asreference and functional test system 23. The reference and functionaltest system differs from the indication system in that its sample andreservoir layer is impregnated with a defined analyte concentration of 1μg/l, so that when applying an appropriate drop of sample or immersingthe sensor into the sample, a defined “minimum signal current” can beexpected at the reference and control test system 22 as a result of theavailable analyte, and the actual measured current will add thereto. Inthe absence of analyte in the sample, said current will be 5 . . . 8 nA.The measured current of the indication system 22 which, depending on theanalyte, is between 0.1 and 20 nA, will decrease by said amount ofminimum signal current. In this way, not only the sensor function duringthe actual measuring procedure can be controlled but also, thereliability of the measurement can be increased by accounting for bothmeasured currents during signal evaluation.

EXAMPLE 4

(FIG. 2)

This example describes an affinity sensor on the basis of the lectinjacalin, which is a glycoprotein (Kumar, G. S., et al., Biosci. 4(1982), 257-61), as selective affine component for the determination ofIgA in blood plasma (Kondoh, H., et al., Immunol. Meth. 88 (1986),171-73). The principal design of the sensor is based on the designdescribed in Example 1. However, freely diffusible IgA-tyrosinaseconjugate was used in layer 12. The tyrosinase (Sigma) was coupled tohuman (plasma) IgA (CALBIOCHEM) in analogy to Example 1. Furthermore,jacalin (Pierce) instead of the scavenger antibody was immobilized inlayers 14 and 16. To this end, Blauband round filter paper (Schleicher &Schull 589/3, diameter: 55 mm) was activated using carbonyldiimidazole(Hissey, P. H. et al., Immunol. Meth. 78 (1985), 211-16), so that thejacalin was covalently bound via NH₂ groups. 3 mg of jacalin in onemilliliter of 0.1 M borate puffer (pH 8.5) was used for a filter paperarea of about 23 cm². For covalent binding, the CDA-activated filterpaper was incubated with the jacalin solution for 24 hours at 8° C. andsubsequently washed with 0.1 M phosphate buffer solution (pH 6.8)containing 2 vol.-% dextran, m.w. 70,000 (Sigma), 10 vol.-% glycerol(Sigma), and dried. Thereafter, the paper was treated with a 1%casein-phosphate buffer/KCl solution (0.1 M), dried, punched out to adiameter of 1 mm, and used as layers 14 and 16 as described in Example1.

When applying a drop of blood plasma to the perforated fixing framecover 8, the sample fluid initially diffuses via spacer 9 into thesample-receiving and reservoir layer 11. As a result of swelling of thesample-receiving and reservoir layer 11 caused in this way, the Siloprenfilm 9 is pressed against the perforated fixing frame cover 8, thusclosing same. The aqueous sample will diffuse through the followingfilter paper layer 12 containing IgA-tyrosinase conjugate, thusmobilizing the conjugate.

After breaking through the hydrophobized paper layer 13 serving asdiffusion barrier, both the conjugate and the analyte enter anotherfilter paper layer 14 containing the jacalin immobilized therein. Duringthe heterogeneous immune reaction, the IgA labelled with tyrosinase andthe IgA of the sample will compete for the binding sites of theimmobilized lectin. Owing to the equilibrium reaction, complexing occursproportionally to the analyte. The more analyte is present in thesample, the less IgA-tyrosinase conjugate will be bound. A sufficientincubation period for the competitive immune reaction is ensured by thehydrophobic paper layer 15 arranged in succession.

Having overcome this diffusion barrier, the non-bound IgA-enzymeconjugate enters the layer 16 in the vicinity of the electrode which, inan identical manner as layer 14, has immobilized lectin so that theIgA-tyrosinase conjugate not bound in layer 14 now is bound by theimmobilized jacalin and thus, is accumulated in front of the electrodesurface 3. Because the sample is an aqueous one, the catechol used asenzyme substrate will be dissolved from the filter paper ring 18surrounding the layer in the vicinity of the electrode, initiating itsenzyme-catalyzed reaction. The catechol is oxidized to o-quinone whichis reduced back to catechol by the cathodically reducedN-methylphenazinium on the electrode surface 3. The cathodic measuredcurrent resulting from this cyclic enzymatic-electrochemical substrateregeneration, which is detected as peak current using square wavevoltammetry at −160 mV versus internal Ag/AgCl pseudo-referenceelectrode 4, is proportional to the IgA concentration.

Reference Index 1 Support 2 Contact paths of the electrodes 3Mediator-modified redox electrode 4 Pseudo-reference electrode 5Isolating layer 6 Fixing frame 7 Fixing frame 8 Top cover of fixingframe, including perforated area 9 Film 10 Spacer 11 Sample-receivingand reservoir layer 12 Layer containing freely diffusible ligand orreceptor labelled with phenol oxidase or hydrolase 13 Diffusion barrierlayer 14 Layer containing an immobilized ligand or receptor 15 Diffusionbarrier layer 16 Layer in the vicinity of the electrode, containing animmobilized ligand or receptor 17 Diffusion barrier layer 18 Enzymesubstrate layer containing a substrate for phenol oxidase 19 Layercontaining immobilized receptor/ligand complexes labelled with phenoloxidase or hydrolase 20 Enzyme substrate layer containing a substratefor the hydrolase 21 Layer in the vicinity of the electrode, containingimmobilized phenol oxidase 22 Measuring system (identical with 23) 23Measuring system (identical with 22)

What is claimed is:
 1. An enzymatic-electrochemical one-step affinitysensor for the quantitative determination of analytes in aqueous media,comprising a support having applied thereon a measuring system or twoadjacent measuring systems of identical design, and the contact pathsthereof, wherein each measuring system comprises multiple consecutivelayers arranged over a redox electrode modified with an electronmediator, and a pseudo-reference electrode, wherein said layers arelaterally encapsulated in a liquid-proof fashion by a fixing framehaving a top cover which has an area having openings to receive thesample to be measured, and wherein said layers comprise 1) either a) alayer including a phenol oxidase substrate, for that case where a markerenzyme of an affine binding partner is a phenol oxidase, or b) a layerincluding a hydrolase substrate and an additional layer in the vicinityof the electrode which includes immobilized phenol oxidase, for thatcase where a marker enzyme of an affine binding partner is a hydrolase,2) a sample-receiving and reservoir layer, 3) a layer in the vicinity ofthe electrode, and 4) a) layers which contain affine binding partners,or b) a layer including appropriate immobilized affinity complexes. 2.The sensor according to claim 1, wherein in the event of two measuringsystems, one measuring system represents an actual indication system,and the other measuring system is used to control calibration arid testthe function of the sensor.
 3. The sensor according to claim 1 or 2,wherein the redox electrode has a polarization voltage of between −300mV and 100 mV versus the pseudo-reference electrode.
 4. The sensoraccording to claim 3, wherein the redox electrode is a mediator-modifiedcarbon electrode and the pseudo-reference electrode is an Ag/AgClelectrode.
 5. The sensor according to claim 1, wherein the electronmediator of the modified redox electrode is fixed on the electrodesurface by adsorption, physical occlusion, covalent bonding, or in theform of a redox polymer.
 6. The sensor according to claim 1, wherein theelectron mediator is a quinoid redox dye, a quinone, a redox-activecomplex compound of iron, ruthenium or tungsten, a metallocene,phthalocyanine, or an electrically conductive redox polymer.
 7. Thesensor according to claim 1, wherein below the top cover of the fixingframe, a water-impermeable film overlapping the perforations of saidcover is arranged, which is spaced apart from said cover by a spacer,and wherein the film is followed by the sample-receiving and reservoirlayer.
 8. The sensor according to claim 7, wherein the water-impermeablefilm is a membrane preferably made of PTFE, polyethylene, polycarbonate,or rubber compounds.
 9. The sensor according to claim 1, whereinfollowing the sample-receiving and reservoir layer, a layer is arrangedwhich either contains a freely diffusible phenol oxidase-labelled ligandor a freely diffusible phenol oxidase-labelled receptor, followed by adiffusion barrier layer and thereafter, a layer having an immobilizedligand or an immobilized receptor is arranged, followed by a seconddiffusion barrier layer which is followed by the reaction layer in thevicinity of the electrode as the last layer, to which either the ligandor receptor is immobilized, and which is enclosed by an enzyme substratelayer which is separated by a diffusion barrier and contains a substratefor the phenol oxidase.
 10. The sensor according to claim 9, wherein ahydrophobized paper is used as one or more of said diffusion barrierlayers.
 11. The sensor according to claim 9, wherein the layerscontaining the affine binding partners, the layers containing the enzymesubstrate, the phenol oxidase layer, or the layer containingenzyme-labelled, immobilized affinity complexes comprise an absorbentmaterial containing a hydrophilic component.
 12. The sensor according toclaim 11, wherein a polysaccharide, a polyalcohol, a poly(etheralcohol), an inorganic salt, or a mixture thereof is used as saidhydrophilic component.
 13. The sensor according to claim 11, whereinsaid hydrophilic component of said absorbent material is a cellulose, apolysilicate, a linear-cross-linked hydrogel or a mixture thereof. 14.The sensor according to claim 1, wherein following the sample-receivingand reservoir layer, a layer is arranged which either contains animmobilized receptor complexed with a phenol oxidase-labelled ligand, oran immobilized ligand complexed with a phenol oxidase-labelled receptor,followed by a diffusion barrier layer which is followed by the reactionlayer in the vicinity of the electrode as the last layer, to whicheither the ligand or receptor is immobilized, and which is enclosed byan enzyme substrate layer which is separated by a diffusion barrier andcontains a substrate for the phenol oxidase.
 15. The sensor according toclaim 1, wherein following the sample-receiving and reservoir layer, alayer is arranged that contains a freely diffusible hydrolase-labelledligand or a freely diffusible hydrolase-labelled receptor, followed by adiffusion barrier layer and thereafter, a layer having an immobilizedligand or an immobilized receptor, followed by a second diffusionbarrier layer, followed by the reaction layer in the vicinity of theelectrode, to which either the ligand or receptor is immobilized, andwhich is enclosed by an enzyme substrate layer that is separated by adiffusion barrier and contains a substrate for the hydrolase, followedby an additional layer in the vicinity of the electrode that is arrangedbetween the reaction layer and redox electrode and that containsimmobilized phenol oxidase.
 16. The sensor according to claim 1, whereinfollowing the sample-receiving and reservoir layer, a layer is arrangedwhich either contains an immobilized receptor complexed with ahydrolase-labelled ligand, or an immobilized ligand complexed with ahydrolase-labelled receptor, followed by a diffusion barrier layer whichis followed by the reaction layer in the vicinity of the electrode, towhich either the ligand or receptor is immobilized, and which isenclosed by an enzyme substrate layer which is separated by a diffusionbarrier and contains a substrate for the hydrolase, followed by anadditional layer in the vicinity of the electrode that is arrangedbetween the reaction layer and redox electrode, and that containsimmobilized phenol oxidase.
 17. The sensor according to claim 1, whereinthe sample-receiving and reservoir layer is capable of swelling andcontains a natural or synthetic hydrogel.
 18. The sensor according toclaim 17, wherein said hydrogel comprises agar, gelatin, pectin,dextrin, polyacrylate or polyurethane.
 19. The sensor according to claim1, wherein tyrosinase is used as said phenol oxidase.
 20. The sensoraccording to claim 19, wherein phenol, m-cresol, p-cresol, 2,4-xylenol,p-chlorophenol, or catechol is used as an enzyme substrate of saidtyrosinase.
 21. The sensor according to claim 1, wherein an alkalinephosphatase, acid phosphatase, or β-galactosidase is used as saidhydrolyzing enzyme.
 22. The sensor according to claim 1, wherein saidelectrically conductive redox polymer is polyaniline, polypyrrole,poly-o-phenylenediame or polyacetylene.
 23. An enzymatic-electrochemicalone-step affinity assay for the quantitative determination of analytesin aqueous media, wherein a sample fluid is applied to a perforated areaof a fixing frame cover of a sensor according to claim 34, which isconnected to a potentiostate or a manual measuring instrument ofpotentiostatic design; and said sample initially, diffuses into thesample-receiving and reservoir layer via the spacer, said layerundergoing swelling, thereby pressing the film against the fixing framecover, thus closing same, and subsequently, the analyte to be determinedin the sample fluid undergoing an affine binding reaction when passingthe layers of the sensor, wherein a) a phenol oxidase-labelled receptor,a phenol oxidase-labelled ligand, or the corresponding phenoloxidase-labelled affinity complex, together with the analyte, diffusesto a mediator-modified electrode surface, and the phenol oxidaseoxidizes a suitable phenol oxidase substrate in the layer in immediatevicinity of the electrode surface to form an electrically active productwhich is reduced cathodically via the reversibly reduced electronmediator to form a starting substrate of the phenol oxidase, or b) aphenol oxidase on a redox electrode is fixed as a layer, and an eductresulting from the reaction of a hydrolyzing enzyme used as label in thesame fashion as in a) with a suitable hydrolase substrate is oxidized inthe immediate vicinity of the electrode surface by the phenol oxidaseused as a catalytic layer to form an electrically active product whichis reduced cathodically via the reversibly reduced electron mediator toform a starting substrate of the phenol oxidase; and the cyclicsubstrate regeneration generated in both cases a) or b) resulting in achemically amplified, analyte-proportional cathodic current which isquantified using per se common voltammetric methods.
 24. The assayaccording to claim 23, wherein the analyte to be determined mobilizesthe phenol oxidase-labelled ligand or receptor a pseudo-homogeneousbinding reaction takes place between the analyte and the diffusiblephenol oxidase-labelled receptor or the phenol oxidase-labelled ligand,the non-bound fraction of the receptor or ligand conjugate, subsequentto overcoming the diffusion barrier layer is bound to immobilized ligandor immobilized receptor, and the analyte receptor conjugate complex orthe analyte-ligand conjugate complex, subsequent to overcoming thediffusion barrier layer, enters the reaction layer in the vicinity ofthe electrode, being bound to an immobilized receptor or ligand, and themarker phenol oxidase, after mobilization of the phenol oxidasesubstrate in the substrate layer and its breaking through the diffusionbarrier layer into the reaction-layer in the vicinity of the electrode,catalyzes an amplifying reaction between the phenol oxidase substrateand the modified electrode surface, thus providing ananalyte-proportional voltammetric current signal.
 25. The assayaccording to claim 23, wherein the analyte to be determined in thesample fluid mobilizes the phenol oxidase-labelled ligand or receptor,the analyte and the phenol oxidase-labelled ligand or the phenoloxidase-labelled receptor, subsequent to overcoming the diffusionbarrier layer, compete with immobilized ligand or receptor for theavailable binding sites, and the non-bound phenol oxidase-labelledligand or phenol oxidase-labelled receptor, subsequent to overcoming thenext diffusion barrier layer, enters the reaction layer in the vicinityof the electrode, being bound to an immobilized receptor or ligand, andthe marker phenol oxidase, after mobilization of the phenol oxidasesubstrate in the substrate layer and its breaking through the diffusionbarrier layer into the reaction layer in the vicinity of the electrode,catalyzes an amplifying reaction between the phenol oxidase substrateand the modified electrode surface, thus providing ananalyte-proportional voltammetric current signal.
 26. The assayaccording to claim 23, wherein the analyte to be determined in thesample fluid reaches layer which contains immobilized ligand orreceptor, the binding sites thereof being saturated with phenoloxidase-receptor conjugate or phenol oxidase ligand conjugate, theanalyte displaces part of the phenol oxidase-ligand conjugate or thephenol oxidase receptor conjugate, and the phenol oxidase ligandconjugate complexed with analyte or the phenol oxidase receptorconjugate complexed with analyte, subsequent to overcoming the diffusionbarrier layer, enters the reaction layer in the vicinity of theelectrode, being bound to the receptor or ligand immobilized therein,and the marker phenol oxidase, after mobilization of the phenol oxidasesubstrate in the substrate layer and its breaking through the diffusionbarrier layer into the reaction layer in the vicinity of the electrode,catalyzes an amplifying reaction between the phenol oxidase substrateand the modified electrode surface, thus providing ananalyte-proportional voltammetric current signal.
 27. The assayaccording to claim 23, wherein the analyte to be determined in thesample fluid mobilizes the hydrolase-labelled receptor or ligand, apseudo-homogeneous binding reaction between the analyte and thediffusible hydrolase-labelled receptor or ligand takes place, thenon-bound fraction of the receptor conjugate or ligand conjugate,subsequent to overcoming the diffusion barrier layer, is bound toimmobilized ligand or receptor in layer, and the analyte-receptorconjugate complex or the analyte-ligand conjugate complex, subsequent toovercoming the diffusion barrier layer, enters the reaction layer in thevicinity of the electrode, being bound to an immobilized receptor orligand, and the marker hydrolase, after mobilization of the hydrolasesubstrate in the substrate layer and its breaking through the diffusionbarrier layer into the reaction layer in the vicinity of the electrode,hydrolyzes an educt which penetrates a layer additionally arranged infront of the electrode surface containing immobilized phenol oxidaseand, being a substrate of phenol oxidase, triggers a phenoloxidase-catalyzed amplifying reaction between the phenol oxidasesubstrate and the modified electrode surface, which provides ananalyte-proportional voltammetric current signal.
 28. The assayaccording to claim 23, wherein the analyte to be determined in thesample fluid mobilizes the hydrolase-labelled receptor or ligand, theanalyte and the hydrolase-labelled ligand or receptor, subsequent toovercoming the diffusion barrier layer, compete with immobilized ligandor immobilized receptor for the binding sites in layer, and thenon-bound fraction of hydrolase-labelled ligand or hydrolase-labelledreceptor, subsequent to overcoming the diffusion barrier layer, entersthe reaction layer in the vicinity of the electrode, being bound to animmobilized receptor or ligand in said reaction layer, and the markerhydrolase, after mobilization of the hydrolase substrate in thesubstrate layer and its breaking through the diffusion barrier layerinto the reaction layer in the vicinity of the electrode, hydrolyzes aneduct which penetrates a layer additionally arranged in front of theelectrode surface containing immobilized phenol oxidase and, being asubstrate of phenol oxidase, triggers a phenol oxidase-catalyzedamplifying reaction between the phenol oxidase substrate and themodified electrode surface, which provides an analyte-proportionalvoltammetric current signal.
 29. The assay according to claim 23,wherein the analyte to be determined in the sample fluid reaches a layerwhich contains immobilized ligand or receptor, the binding sites thereofbeing saturated with hydrolase-receptor conjugate or hydrolase-ligandconjugate, the analyte displaces part of the hydrolase-ligand conjugateor the hydrolase-receptor conjugate, and the hydrolase-ligand conjugatecomplexed with analyte or the hydrolase-receptor conjugate complexedwith analyte, subsequent to overcoming the diffusion barrier layer,enters the reaction layer in the vicinity of the electrode, being boundto the receptor or ligand immobilized therein, and the marker hydrolase,after mobilization of the hydrolase substrate in the substrate layer andits breaking through the diffusion barrier layer into the reaction layerin the vicinity of the electrode, hydrolyzes an educt which penetrates alayer additionally arranged in front of the electrode surface containingimmobilized phenol oxidase and, being a substrate of phenol oxidase,triggers a phenol oxidase-catalyzed amplifying reaction between thephenol oxidase substrate and the modified electrode surface, whichprovides an analyte-proportional voltammetric current signal.
 30. Amethod of determining chemical affinity comprising using a hydrolyzingphenolic compound as a marker enzyme for affine binding partners, and aphenol oxidase as a catalyst for an amplifying reaction between a phenoloxidase substrate and a redox mediator and determining the bond formedbetween affine partners in an electrochemical assay.
 31. The method ofclaim 30, further comprising employing a phosphatase or galactosidase assaid hydrolyzing enzyme, and tyrosinase as said phenol oxidase.